Demineralized bone matrix having improved handling characteristics

ABSTRACT

Provided is an injectable implant configured to fit at or near a bone defect to promote bone growth, the injectable implant comprising lyophilized demineralized bone matrix (DBM) being in fiber and particle forms; alginate; and a liquid carrier, wherein the DBM is in an amount of about 20 wt. % to about 40 wt. % of a total weight of the injectable implant, the alginate is in an amount of from about 3 wt. % to about 10 wt. % of the total weight of the injectable implant, and the liquid carrier is in an amount from about 50 wt. % to about 70 wt. % of the total weight of the injectable implant. A moldable implant and methods of making the implants are further provided.

BACKGROUND

The rapid and effective repair of bone defects caused by injury,disease, wounds, or surgery is a goal of orthopedic surgery. Toward thisend, a number of compositions and materials have been used or proposedfor use in the repair of bone defects. The biological, physical, andmechanical properties of the compositions and materials are among themajor factors influencing their suitability and performance in variousorthopedic applications.

Autologous bone (“ACB”), also known as autograft or autogenous bone, isconsidered the gold standard for bone grafts. Autograft bone isosteoinductive and nonimmunogenic, and, by definition, has all of theappropriate structural and functional characteristics appropriate forthe particular recipient. Unfortunately, autograft bone is onlyavailable in a limited number of circumstances. Some individuals lackautograft bone of appropriate dimensions and quality fortransplantation, and donor site pain and morbidity can pose seriousproblems for patients and their physicians. Among the known bone repairmaterials and bone void fillers is autologous cancellous bone. This typeof bone has the advantage of being both osteoinductive andnon-immunogenic. Unfortunately, this type of bone is not available underall circumstances.

Moreover, donor site morbidity and trauma add to the limitations ofautologous cancellous bone. One alternative to autologous bone isallograft bone. Unfortunately, allograft bone has a lower osteogeniccapacity than autograft bone, has a high resorption rate, creates lessrevascularization at the bone defect site, typically induces a greaterimmunogenic response and may result in the transfer of certain diseases.

Much effort has been invested in the identification or development ofalternative bone graft materials. Demineralized bone matrix (“DBM”)implants have been reported to be particularly useful. Demineralizedbone matrix is typically derived from cadavers. The bone is removedaseptically and/or treated to kill any infectious agents. The bone isthen optionally particulated by milling or grinding. The bone is thentreated to remove fats and to extract the mineral components forexample, by soaking the bone in an acidic solution.

DBM is a desirable component of bone graft materials because it providesan osteoinductive matrix and exhibits osteoconductive potential, therebypromoting bone growth and healing. DBM is osteoinductive due to thepresence of active bone growth factors including bone morphogeneticproteins (BMP). Osteoinductivity depends not only on the concentrationof growth factors in DBM, but also on their availability to cells afterimplantation. Moreover, DBM is fully resorbable, and bone graftmaterials containing organic DBM are highly biocompatible because itcontains many of the components of natural bone. Following implantation,the presence of DBM induces cellular recruitment to the site of injury.The recruited cells may eventually differentiate into bone formingcells. Such recruitment of cells leads to an increase in the rate ofwound healing and, therefore, to faster recovery for the patient.Advantageously, DBM costs less than many other available organic bonecomposition additives, such as isolated bone morphogenetic proteins(BMPs).

One form of administering DBM is by employing solid bone grafts.However, solid bone grafts cannot be effectively manipulated intoirregularly shaped bone voids or defects and are at times not ideal forgraft placement. DBM putty is therefore beneficial when there is a needto administer a bone graft to irregularly shaped bone voids or defects.Some advantages of using DBM putty is that the putty is moldable and canbe easily manipulated into a bone void or defect site. Further, DBMputty can be administered via injection and can be conveniently storedin syringes, thus preventing the DBM putty from drying out andcontamination when not in use.

Another component that is often found in bone graft materials since itassists in osteoconduction and osteoinduction of bone is collagen.However, at times, patients can have an adverse reaction to collagen,especially collagen that is derived from a non-human source. Forexample, an immunogenic reaction can occur in patients when non-humancollagen is used. An immunogenic reaction is the ability of a particularsubstance to provoke an immune response in the body. In other words, itis the ability to induce a humoral and/or cell-mediated immune response.

Further, bone graft materials are commonly irradiated for sterilizationpurposes. However, sterilization can stripe away natural properties ofbone, for example, by denaturing proteins such as natural collagen,which decreases new bone formation via osteoconduction andosteoinduction.

Therefore, there is a need for implantable devices/carriers comprisingDBM that are injectable and/or moldable and are configured to fit intomany types of bone void defects (e.g., regular or irregular shapeddefects). There is also a need for aseptically processed implantabledevices/carriers that have enhanced osteoinductivity andosteoconductivity. Accordingly, there is a need for implantabledevice/carriers that provide high performance and handlingcharacteristics for implantation into a bone void defect.

SUMMARY

An injectable implant is provided that is configured to fit at or near abone defect to promote bone growth. The injectable implant provides highperformance and handling characteristics for implantation into a bonevoid defect. The injectable implant comprises lyophilized demineralizedbone matrix (DBM) being in fiber and particle forms; alginate; and aliquid carrier. The DBM is in an amount of about 20 wt. % to about 40wt. % of a total weight of the injectable implant, the alginate is in anamount of from about 1 wt. % to about 10 wt. % of the total weight ofthe injectable implant, and the liquid carrier is in an amount fromabout 50 wt. % to about 70 wt. % of the total weight of the injectableimplant.

In some embodiments, a moldable implant is provided that is configuredto fit at or near a bone defect to promote bone growth. The moldableimplant comprises lyophilized demineralized bone matrix (DBM) being infiber, particle and chip forms. The DBM is in an amount of about 15 wt.% to about 40 wt. % based on a total weight of the moldable implant. Themoldable implant further comprises an alginate in an amount of fromabout 3 wt. % to about 20 wt. % based on the total weight of themoldable implant.

In some embodiments, a method of making an injectable or moldableimplant is provided, the method comprising mixing lyophilizeddemineralized bone matrix (DBM) being in fiber and particle forms in anamount of about 28% based on a total weight of the implant, or DBM infiber, particle and chip forms in an amount of about 30% based on thetotal weight of the implant with an aqueous liquid comprising analginate so as to uniformly distribute the DBM within the alginate toform the injectable or moldable implant.

Additional features and advantages of various embodiments will be setforth in part in the description that follows, and in part will beapparent from the description, or may be learned by practice of variousembodiments. The objectives and other advantages of various embodimentswill be realized and attained by means of the elements and combinationsparticularly pointed out in the description and appended claims.

BRIEF DESCRIPTION OF THE FIGURES

In part, other aspects, features, benefits and advantages of theembodiments will be apparent with regard to the following description,appended claims and accompanying drawings where:

FIG. 1 illustrates an injectable implant configured to increaseosteoinductivity and osteoconductivity in bone, the injectable implantcomprising DBM fibers, DBM particles, alginate, phosphate bufferedsaline and sterile filtered water.

FIG. 2 illustrates a moldable implant configured to increaseosteoinductivity and osteoconductivity in bone, the moldable implantcomprising DBM fibers, DBM particles, DBM chips, alginate, phosphatebuffered saline, and sterile filtered water.

FIG. 3 is a table of results from a rat two-level posterior lateralfusion (PLF) study where subjects were divided into two groups; group 1,where an injectable aseptic implant was implanted into the subjects at asurgical site and group 2, where an irradiated moldable putty wasimplanted into the subjects at a surgical site. Results showed that theirradiated moldable putty fused 8 of 24 unilateral segments (33%) whilethe injectable aseptic implant fused 2.4 of 24 unilateral segments(100%) by manual palpation and radiographically. Further, greaterconsolidation and organization of new bone was observed radiographicallyin the aseptic vs. irradiated treatment groups.

FIG. 4 illustrates an x-ray of rat subjects in group 1 that wereadministered the injectable aseptic implant and their progress at 2weeks, 4 weeks, and 8 weeks after implantation.

FIG. 5 illustrates an x-ray of rat subjects from group 2 that wereadministered the irradiated moldable putty and their progress at 2weeks, 4 weeks, and 8 weeks after implantation,

FIG. 6 illustrates a table of results from a rabbit PLF study wheresubjects were divided into 6 groups; Group 1 contained subjects thatwere implanted with an injectable putty at a surgical site, Group 2contained subjects that were implanted with a moldable implant at asurgical site, Group 3 contained subjects that were implanted with acombination product comprising the injectable putty of Group 1 and 50%autograft at a surgical site, Group 4 contained subjects that wereimplanted with a combination product comprising the moldable implant ofGroup 2 and 50% autograft at a surgical site, Group 5 contained subjectsthat were implanted with the predicate irradiated putty at a surgicalsite, and Group 6 contained subjects that were implanted with thecontrol autograft at a surgical site. Results showed that Group 1 causedbilateral fusion in 3 out of 6 segments and unilateral fusion in 6 outof 12 segments, Group 2 caused bilateral fusion in 0 out of 6 segmentsand unilateral fusion in 1 out of 12 segments, Group 3 caused bilateralfusion in 3 out of 6 segments and unilateral fusion in 6 out of 12segments, Group 4 caused bilateral fusion in 1 out of 6 segments andunilateral fusion in 2 out of 12 segments, Group 5 caused bilateralfusion in 0 out of 6 segments and unilateral fusion in 0 out of 12segments, and Group 6 caused bilateral fusion in 3 out of 6 segments andunilateral fusion in 6 out of 12 segments.

FIG. 7 illustrates an x-ray of rabbit subjects in Group 1 that wereadministered the injectable putty.

FIG. S illustrates an x-ray of rabbit subjects in Group 2 that wereadministered the moldable implant.

It is to be understood that the figures may not be to scale. Further,the relationship between objects in a figure may not be to scale, andmay in fact have a reverse relationship as to size. The figures areintended to bring understanding and clarity to the structure of eachobject shown, and thus, some features may be exaggerated in order toillustrate a specific feature of a structure.

DETAILED DESCRIPTION

For the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing quantities of ingredients,percentages or proportions of materials, reaction conditions, and othernumerical values used in the specification and claims, are to beunderstood as being modified in all instances by the term “about.”Accordingly, unless indicated to the contrary, the numerical parametersset forth in the following specification and attached claims areapproximations that may vary depending upon the desired propertiessought to be obtained by the present application. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical parameter shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the present application are approximations, thenumerical values set forth in the specific examples are reported asprecisely as possible. Any numerical value, however, inherently containscertain errors necessarily resulting from the standard deviation foundin their respective testing measurements. Moreover, all ranges disclosedherein are to be understood to encompass any and all sub ranges subsumedtherein. For example, a range of “1 to 10” includes any and all subranges between (and including) the minimum value of 1 and the maximumvalue of 10, that is, any and all sub ranges having a minimum value ofequal to or greater than 1 and a maximum value of equal to or less than10, e.g., 5.5 to 10.

Definitions

It is noted that, as used in this specification and the appended claims,the singular forms “a,” “an,” and “the,” include plural referents unlessexpressly and unequivocally limited to one referent. Thus, for example,reference to “an implant” includes one, two, three or more implants.

The term “bioactive agent” as used herein is generally meant to refer toany substance that alters the physiology of a patient. The term“bioactive agent” may be used interchangeably herein with the terms“therapeutic agent,” “therapeutically effective amount,” and “activepharmaceutical ingredient”, “API” or “drug”.

Bioactive agent or bioactive compound is used herein to refer to acompound or entity that alters, inhibits, activates, or otherwiseaffects biological or chemical events. For example, bioactive agents mayinclude, but are not limited to, osteogenic or chondrogenic proteins orpeptides, anti-AIDS substances, anti-cancer substances, antibiotics,immunosuppressants, anti-viral substances, enzyme inhibitors, hormones,neurotoxins, opioids, hypnotics, anti-histamines, lubricants,tranquilizers, anti-convulsants, muscle relaxants and anti-Parkinsonsubstances, anti-spasmodics and muscle contractants including channelblockers, miotics and anti-cholinergics, anti-glaucoma compounds,anti-parasite and/or anti-protozoal compounds, modulators ofcell-extracellular matrix interactions including cell growth inhibitorsand antiadhesion molecules, vasodilating agents, inhibitors of DNA, RNAor protein synthesis, anti-hypertensives, analgesics, anti-pyretics,steroidal and non-steroidal anti-inflammatory agents, anti-angiogenicfactors, angiogenic factors, anti-secretory factors, anticoagulantsand/or antithrombotic agents, local anesthetics, prostaglandins,anti-depressants, anti-emetics, and imaging agents. In certainembodiments, the bioactive agent is a drug. Bioactive agents furtherinclude RNAs, such as siRNA, and osteoclast stimulating factors. In someembodiments, the bioactive agent may be a factor that stops, removes, orreduces the activity of bone growth inhibitors. In some embodiments, thebioactive agent is a growth factor, cytokine, extracellular matrixmolecule or a fragment or derivative thereof, for example, a cellattachment sequence such as RGD. A more complete listing of bioactiveagents and specific drugs suitable for use in the present applicationmay be found in “Pharmaceutical Substances: Syntheses, Patents,Applications” by Axel Kleemann and Jurgen Engel, Thieme MedicalPublishing, 1999; the “Merck Index: An Encyclopedia of Chemicals, Drugs,and Biologicals”, edited by Susan Budavari et al., CRC Press, 1996; andthe United States Pharmacopeia-25/National Formulary-20, published bythe United States Pharmacopeia Convention, Inc., Rockville Md., 2001,each of which is incorporated herein by reference.

Biocompatible, as used herein, is intended to describe materials that,upon administration in vivo, do not induce undesirable long-termeffects.

The term “biodegradable” includes compounds or components that willdegrade over time by the action of enzymes, by hydrolytic action and/orby other similar mechanisms in the human body. In various embodiments,“biodegradable” includes that components can break down or degradewithin the body to non-toxic components as cells (e.g., bone cells)infiltrate the components and allow repair of the defect. By“bioerodible” it is meant that the compounds or components will erode ordegrade over time due, at least in part, to contact with substancesfound in the surrounding tissue, fluids or by cellular action. By“bioabsorbable” it is meant that the compounds or components will bebroken down and absorbed within the human body, for example, by a cellor tissue. “Biocompatible” means that the compounds or components willnot cause substantial tissue irritation or necrosis at the target tissuesite and/or will not be carcinogenic.

A “therapeutically effective amount” or “effective amount” is such thatwhen administered, the bioactive agent results in alteration of thebiological activity, such as, for example, enhancing bone growth, etc.The dosage administered to a patient can be as single or multiple dosesdepending upon a variety of factors, including the drug's administeredpharmacokinetic properties, the route of administration, patientconditions and characteristics (sex, age, body weight, health, size,etc.), and extent of symptoms, concurrent treatments, frequency oftreatment and the effect desired. In some embodiments the formulation isdesigned for immediate release. In other embodiments the formulation isdesigned for sustained release. In other embodiments, the formulationcomprises one or more immediate release surfaces and one or moresustained release surfaces.

A “depot” includes but is not limited to capsules, microspheres,microparticles, microcapsules, microfibers particles, nanospheres,nanoparticles, coating, matrices, wafers, pills, pellets, emulsions,liposomes, micelles, gels, or other pharmaceutical delivery compositionsor a combination thereof. Suitable materials for the depot are ideallypharmaceutically acceptable biodegradable and/or any bioabsorbablematerials that are preferably FDA approved or GRAS materials. Thesematerials can be polymeric or non-polymeric, as well as synthetic ornaturally occurring, or a combination thereof. In some embodiments, theimplant can be a biodegradable depot.

The term “implantable” as utilized herein refers to a biocompatibledevice (e.g., implant) retaining potential for successful placementwithin a mammal. The expression “implantable device” and expressions ofthe like import as utilized herein refers to an object implantablethrough surgery, injection, or other suitable means whose primaryfunction is achieved either through its physical presence or mechanicalproperties.

“Localized” delivery includes delivery where one or more implants aredeposited within a tissue, for example, a bone cavity, or in closeproximity (within about 0.1 cm, or preferably within about 10 cm, forexample) thereto.

The term “immunogenic,” “immunogenic reaction,” or “immunogenicity”refers to the ability of a particular substance, such as an antigen orepitope, to provoke an immune response in the body of a human or animal.In other words, immunogenicity is the ability to induce a humoral and/orcell-mediated immune response. “Unwanted immunogenicity” refers to animmune response by an organism against a therapeutic antigen (e.g.,recombinant protein, or monoclonal antibody). This reaction leads toproduction of anti-drug-antibodies (ADAs) inactivating the therapeuticeffects of the treatment and, in rare cases, inducing adverse effects.

The term “mammal” refers to organisms from the taxonomy class“mammalian,” including but not limited to humans, other primates such asmonkeys, chimpanzees, apes, orangutans and monkeys, rats, mice, rabbits,cats, dogs, pigs, cows, horses, etc.

The term “particle” refers to pieces of DBM bone material having variousshapes and sizes that possess regular, irregular or random geometries.In some embodiments. DBM particles include shapes, such as for example,spheres, cubes, cylinders, ovals, granular, or the like. It should beunderstood that some variation in dimension will occur in the productionof the particles and particles demonstrating such variability indimensions are within the scope of the present application. Particles donot include fibers and chips.

In some embodiments, the implant comprises a matrix. The “matrix” of thepresent application is utilized as a scaffold for bone and/or cartilagerepair, regeneration, and/or augmentation. Typically, the matrixprovides a 3-D matrix of interconnecting pores, which acts as a scaffoldfor cell migration. The morphology of the matrix guides cell migrationand cells are able to migrate into or over the matrix, respectively. Thecells then are able to proliferate and synthesize new tissue and formbone and/or cartilage. In some embodiments, the matrix can be a putty,paste, cohesive mass, or injectable form.

In some embodiments, the implant can be malleable, cohesive, flowableand/or can be shaped into any shape. The term “malleable” includes thatthe implant is capable of being converted from a first shape to a secondshape by the application of pressure, such as, for example, a putty.

The term “cohesive” as used herein means that the implant tends toremain a singular, connected mass upon movement, including theexhibition of the ability to elongate substantially without breakingupon stretching. An example of a cohesive implant includes, for example,a putty.

The term “moldable” includes that the implant can be shaped by hand ormachine or injected in the target tissue site (e.g., bone defect,fracture, or void) into a wide variety of configurations. In someembodiments, the implant can be formed into sheets, blocks, rings,struts, plates, disks, cones, pins, screws, tubes, teeth, bones, portionof bone, wedges, cylinders, threaded cylinders, or the like, as well asmore complex geometric configurations.

The term “injectable” refers to a mode of administering the implant. Theimplant can be administered in a variety of ways such as, for example, asyringe and/or cannula. For example, the implant can be administeredparenterally, such as for example, anterior lumbar interbodyadministration for fusion, or posterior lumbar interbody administrationfor fusion or transforaminal lumbar interbody administration for fusion,other intraspinal injection or other local administration.

The term “alginate” or “alginates” refer to a type of polysaccharideisolated from brown algae. It is a linear copolymer with homopolymericblocks of guluronic (G) and mannuronic (M) acids as G monomer blocks, Mmonomer blocks and guluronic-triannuronic (G-M) alternating sequences.Among these three types of structural elements, only G blocks areinvolved in the gelation of alginate by reacting with multiple cationssuch as Ca²⁺ and Ba²⁺.

The monomers can appear in homopolymeric blocks of consecutiveG-residues (G-blocks), consecutive M-residues (M-blocks), alternating Mand G-residues (G-M blocks) or randomly organized blocks. The relativeamount of each block type varies with the origin of the alginate.Alternating blocks form the most flexible chains and are more soluble atlower pH than the other blocks. G-blocks form stiff chain elements, andtwo G-blocks of more than 6 residues each can form ionicallycross-linked junctions with divalent cations e.g. Ca²⁺, Ba²⁺, Sr²⁻ amongothers, leading to a three-dimensional gel network. In these ionicallycross-linked gels, it is mostly the homopolymeric G blocks that form thejunctions, where the stability of the gel is determined by the relativeamount of divalent cations combined.

There exists a free carboxyl group on each of the plutonic or mannuronicmoiety. When these carboxyl groups are not ionized, e.g. in a low pHaqueous environment, alginate molecules are not hydrated and becomeinsoluble. However, alginate molecules become soluble and fully hydratedwhen the pH is neutral or alkaline. Under this condition, the reactionbetween G blocks and cations such as Ca²⁺ and Ba²⁻ leads to the ionicgelation of alginate.

In some embodiments, the alginate comprises sodium alginate, potassiumalginate, calcium alginate, ammonium alginate, propylene glycolalginate, or a combination thereof.

The term “aseptic”, refers to an implant that is free from contaminationcaused by bacteria, viruses, or other microorganisms.

Bone, as used herein, refers to bone that is cortical, cancellous orcortico-cancellous of autogenous, allogenic, xenogenic, or transgenicorigin.

Bone graft, as used herein, refers to any implant prepared in accordancewith the embodiments described herein and therefore may includeexpressions such as bone material and bone membrane.

Demineralized, as used herein, refers to any material generated byremoving mineral material from tissue, for example, bone tissue. Incertain embodiments, the demineralized compositions described hereininclude preparations containing less than 5% calcium. In someembodiments, the demineralized compositions may comprise less than 1%calcium by weight. In some embodiments, the compositions may compriseless than 5, 4, 3, 2 and/or 1% calcium by weight. Partiallydemineralized bone is intended to refer to preparations with greaterthan 5% calcium by weight but containing less than 100% of the originalstarting amount of calcium. In some embodiments, partially demineralizedbone comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 3738, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49. 50, 51, 52, 53, 55, 56.57, 58, 59, 60, 61, 62, 63, 64, 65. 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98 and/or 99% of the original starting amount ofcalcium.

In some embodiments, demineralized bone has less than 95% of itsoriginal mineral content. In some embodiments, demineralized bone lessthan 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80. 79,78, 77, 76, 75, 74, 73. 72, 71, 70, 69, 68, 67, 66, 65, 64. 63, 62, 61,60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43,42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25,24. 23, 22, 21. 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6and/or 5% of its original content. In some embodiments, “Demineralized”is intended to encompass such expressions as “substantiallydemineralized,” “partially demineralized,” “surface demineralized,” and“fully demineralized.” “Partially demineralized” is intended toencompass “surface demineralized.”

In some embodiments, the demineralized bone may be surface demineralizedfrom about 1-99%. In some embodiments, the demineralized bone is 1, 2,3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 7 8 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98 and/or 99% surface demineralized. In various embodiments,the demineralized bone may be surface demineralized from about 15-25%.In some embodiments, the demineralized bone is 15, 16, 17, 18, 19, 20,21, 22, 23, 24 and/or 25% surface demineralized.

Demineralized bone matrix (DBM), as used herein, refers to any materialgenerated by removing mineral material from bone tissue. In someembodiments, the DBM compositions as used herein include preparationscontaining less than 5% calcium and, in some embodiments, less than 1%calcium by weight. In some embodiments, the DBM compositions includepreparations that contain less than 5, 4, 3, 2 and/or 1% calcium byweight. In other embodiments, the DBM compositions comprise partiallydemineralized bone (e.g., preparations with greater than 5% calcium byweight but containing less than 100% of the original starting amount ofcalcium).

In some embodiments, part or all of the surface of the bone can bedemineralized. For example, part or all of the surface of the bonematerial can be demineralized to a depth of from about 100 to about 5000microns, or about 150 microns to about 1000 microns. In someembodiments, part or all of the surface of the bone material can bedemineralized to a depth of from about 100, 150, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050,1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650,1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250,2300, 2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850,2900, 2950, 3000, 3050, 3100, 3150, 3200, 3250, 3300, 3350, 3400, 3450,3500, 3550, 3600, 3650, 3700, 3750, 3800, 3850, 3900, 3950, 4000, 4050,4100, 4150, 4200, 4250, 4300, 4350, 4400, 4450, 4500, 4550, 4600, 4650,4700, 4750, 4800, 4850, 4900, 4950 to about 5000 microns. If desired,the implant can comprise demineralized material.

Osteoconductive, as used herein, refers to the ability of a substance toserve as a template or substance along which bone may grow.

Osteogenic, as used herein, refers to materials containing living cellscapable of differentiation into bone tissue.

Osteoinductive, as used herein, refers to the quality of being able torecruit cells from the host that have the potential to stimulate newbone formation. Any material that can induce the formation of ectopicbone in the soft tissue of an animal is considered osteoinductive. Forexample, most osteoinductive materials induce bone formation in athymicrats when assayed according to the method of Edwards et al.,“Osteoinduction of Human Demineralized Bone: Characterization in a RatModel,” Clinical Orthopaedics & Rel. Res., 357:219-228, December 1998,incorporated herein by reference.

Superficially demineralized, as used herein, refers to bone-derivedelements possessing at least about 90 weight percent of their originalinorganic mineral content. In some embodiments, superficiallydemineralized contains at least about 90, 91, 92, 93, 94, 95, 96, 97, 98and/or 99 weight percent of their original inorganic material. Theexpression “partially demineralized” as used herein refers tobone-derived elements possessing from about 8 to about 90 weight percentof their original inorganic mineral content. In some embodiments,partially demineralized contains about 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89 and/or 90 weight percent of their original inorganic mineral content.The expression “fully demineralized” as used herein refers to bonecontaining less than 8% of its original mineral context. In someembodiments, fully demineralized contains about less than 8, 7, 6, 5, 4,3, 2 and/or 1% of its original mineral content.

The expression “average length to average thickness ratio” as applied tothe DBM fibers of the present application means the ratio of the longestaverage dimension of the fiber (average length) to its shortest averagedimension (average thickness). This is also referred to as the “aspectratio” of the fiber.

Fibrous, as used herein, refers to bone elements whose average length toaverage thickness ratio or aspect ratio of the fiber is from about 50:1to about 1000:1. In some embodiments, average length to averagethickness ratio or aspect ratio of the fiber is from about 50:1, 75:1,100:1, 125:1, 150:1, 175:1, 200:1, 225:1, 250:1, 275:1, 300:1, 325:1,350:1, 375:1, 400:1, 425:1, 450:1, 475:1, 500:1, 525:1, 550:1, 575:1,600:1, 625:1, 650:1, 675:1, 700:1, 725:1, 750:1, 775:1, 800:1, 825:1,850:1, 875:1, 900:1, 925:1, 950:1, 975:1 to about 1000:1. In overallappearance the fibrous bone elements can be described as bone fibers,threads, narrow strips, or thin sheets. Often, where thin sheets areproduced, their edges tend to curl up toward each other. The fibrousbone elements can be substantially linear in appearance or they can becoiled to resemble springs. In some embodiments, the bone fibers are ofirregular shapes including, for example, linear, serpentine or curvedshapes. The bone fibers are preferably demineralized however some of theoriginal mineral content may be retained when desirable for a particularembodiment. In various embodiments, the bone fibers are mineralized. Insome embodiments, the fibers are a combination of demineralized andmineralized.

Non-fibrous, as used herein, refers to elements that have an averagewidth substantially larger than the average thickness of the fibrousbone element or aspect ratio of less than from about 50:1 to about1000:1. Preferably the non-fibrous bone elements are shaped in asubstantially regular manner or specific configuration, for example,triangular prism, sphere, cube, cylinder and other regular shapes. Bycontrast, particles such as chips, shards, or powders possess irregularor random geometries. It should be understood that some variation indimension will occur in the production of the elements of thisapplication and elements demonstrating such variability in dimension arewithin the scope of this application and are intended to be understoodherein as being within the boundaries established by the expressions“mostly irregular” and “mostly regular”.

“Surface demineralized fibrous bone chip,” as used herein, refers tosurface demineralized bone chip(s) that have been subjected to mildpressure from about 5,000, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000,8,500, 9,000, 9,500, to about 10,000 pounds per square inch (psi).

The section headings below should not be restricted and can beinterchanged with other section headings.

Implantable Device

In some embodiments, an injectable implant 10 is provided that isconfigured to fit at or near a bone defect to promote bone growth, asshown in FIG. 1. In some embodiments, the injectable implant isadministered via a syringe and/or a cannula. In sonic embodiments, thebone defect is a bone void. The injectable implant is configured to be abone void filler that is injectable in a putty form. The injectableimplant comprises lyophilized demineralized bone matrix (DBM) 12 infiber and particle forms, alginate 14, and a liquid carrier such asphosphate buffered saline (PBS) 16. The injectable implant shown in 10has a combination of DBM fibers and DBM particles that are uniformlydistributed throughout the alginate. The injectable implant also doesnot contain collagen so that an immunogenic reaction does not occur inpatients that are administered the injectable implant.

The injectable implant is aseptically processed to enhanceosteoinductivity and osteoconductivity of bone. Aseptically processingtechniques as described below, prevent natural properties of bone frombeing striped, thus increasing the production of new bone formation viaosteoconduction and osteoinduction.

In some embodiments, the DBM is fully demineralized. In someembodiments, the fully demineralized DBM contains about less than 8, 7,6, 5, 4, 3, 2 and/or 1% of its original mineral content. In someembodiments, the DBM is partially demineralized, surface demineralizedand/or fully demineralized.

In some embodiments, the DBM is in both fiber and particle forms. Insome embodiments, the fibers have a size of from about 1 to about 7 mm.In some embodiments, the fibers have a size from about 1 to about 20 mm,from about 1 to about 15 mm, from about 1 to about 10 mm, from about 2to about 15 mm, from about 2 to about 10 mm, from about 2 to about 5 mm,from about 3 to about 15 mm, from about 3 to about 10 mm, from about 1to about 5 mm, from about 4 to about 15 mm, from about 10 mm, from about4 to about 5 mm, from about 5 to about 15 mm, or from about 5 to about10 mm. In some embodiments, the fibers have a size from about 1 to about50 mm, from about 5 to about 40 mm, from about 5 to about 30 mm, fromabout 5 to about 10 mm. In some embodiments, the fibers have a size offrom about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 to about 50 mm,

In some embodiments, the particles have a size of from about 100 micronsto about 1000 microns. In some embodiments, the particles have a size offrom about 100 microns to about 500 microns, from about 100 microns toabout 250 microns, from about 200 microns to about 1000 microns, fromabout 200 microns to about 800 microns, from about 200 microns to about600 microns, from about 200 microns to about 400 microns, from about 200microns to about 300 microns, from about 300 microns to about 800microns, from about 300 microns to about 600 microns, from about 300microns to about 400 microns, from about 400 microns to about 1000microns, from about 400 microns to about 600 microns, from about 500microns to about 1000 microns, from about 500 microns to about 800microns, from about 500 microns to about 600 microns.

In some embodiments, the particles have a size of from about 1, 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,105, 11.0, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170,175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240,245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310,315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380,385, 390, 395, 400, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445,450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515,520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585,590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655,660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725,730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795,800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865,870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935,940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995 to about 1000microns.

In some embodiments, the DBM is in an amount of about 20 wt. % to about40 wt. % of a total weight of the injectable implant. In someembodiments, the DBM is in an amount of from about 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, to about 40wt. % of the total weight of the implant. In some embodiments, the DBMis in an amount of about 1 to about 99 wt. % of the total weight of theimplant. In some embodiments, the DBM is in an amount of from about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, to about 99 wt. % of the total weight of the implant.

In some embodiments, the injectable implant comprises a ratio of fiberand particle forms of about 50:50. In some embodiments, the injectableimplant comprises a ratio of fibers and particle forms of from about50:1, 1:50, 25:1, 1:25, 75:25, 25:75, 1:12, 12:1, 1:10, 10:1, 8:1, 1:8,6:1, 1:6, 5:1, 1:5, or 4:1 to about 1:4,

In some embodiments, from about 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, to about 99% of theDBM comprises the fiber form (e.g., fibers) In some embodiments, about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, or 99 of the DBM comprises the particle form(e.g., particles).

In some embodiments, the alginate is in an amount of from about 1 wt. %to about 10 wt. % of the total weight of the injectable implant. In someembodiments, the alginate is in an amount of from about 3 to about 8 wt.%, of from about 5 to about 7 wt. % or of from about 4 to 6 wt. % of thetotal weight of the injectable implant, In some embodiments, thealginate is in an amount of from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, to about99 wt. % of the total weight of the implant.

In some embodiments, the alginate is a sterile sodium alginate. In someembodiments, the alginate is sodium alginate, potassium alginate,calcium alginate, ammonium alginate, propylene glycol alginate, or acombination thereof.

In some embodiments, the alginate is in a powder form and has a particlesize of from about 100 to about 1,000 microns. In some embodiments, theparticle size of the alginate is of from about 100 microns to about 500microns, from about 100 microns to about 250 microns, from about 200microns to about 1000 microns, from about 200 microns to about 800microns, from about 200 microns to about 600 microns, from about 200microns to about 400 microns, from about 200 microns to about 300microns, from about 300 microns to about 800 microns, from about 300microns to about 600 microns, from about 300 microns to about 400microns, from about 400 microns to about 1000 microns, from about 400microns to about 600 microns, from about 500 microns to about 1000microns, from about 500 microns to about 800 microns, from about 500microns to about 600 microns.

In some embodiments, the alginate powder has a particle size of fromabout 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155,160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225,230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295,300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365,370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435,440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505,510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575,580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645,650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715,720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785,790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855,860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925,930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995 toabout 1000 microns.

In some embodiments, the alginate has an average molecular weight ofabout 100,000 to about 600,000 Daltons (Da). In some embodiments, thealginate has an average molecular weight of from about 100,000 to about500,000 Da, or of from about 200,000 to about 400,000 Da. In someembodiments, the alginate has an average molecular weight of from about200,000, 225,000, 250,000, 275,000, 300,000, 325,000, 350,000, 375,000,400,000, 425,000, 450,000, 475,000, 500,000 525,000, 550,000, 575,000 orabout 600,000 Da.

In some embodiments, when the alginate powder is mixed in a solution,such as the phosphate buffered saline, the alginate comprises aviscosity of about 20 to about 2,000 centipoises (cps). In someembodiments, the alginate comprises a viscosity of from about 20, 30,40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320,330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460,470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600,610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740,750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880,890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020,1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140,1150, 1160, 1170, 1180, 1190, 1200, 1210, 1220, 1230, 1240, 1250, 1260,1270, 1280, 1290, 1300, 1310, 1320, 1330, 1340, 1350, 1360, 1370, 1380,1390, 1400, 1410, 1420, 1430, 1440, 1450, 1460, 1470, 1480, 1490, 1500,1510, 1520, 1530, 1540, 1550, 1560, 1570, 1580, 1590, 1600, 1610, 1620,1630, 1640, 1650, 1660, 1670, 1680, 1690, 1700, 1710, 1720, 1730, 1740,1750, 1760, 1770, 1780, 1790, 1800, 1810, 1820, 1830, 1840, 1850, 1860,1870, 1880, 1890, 1900, 1910, 1920, 1930, 1940, 1950, 1960, 1970, 1980,1990 to about 2000 cps.

In some embodiments, the phosphate buffered saline is in an amount fromabout 50 wt. % to about 70 wt. % of the total weight of the injectableimplant. In some embodiments, the phosphate buffered saline is in anamount of from about 55 wt. % to about 65 wt. %, or from about 60 wt. %)to about 70 %) of the total weight of the injectable implant. In someembodiments, the phosphate buffered saline is in an amount of 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,or 70 wt. % of the total weight of the injectable implant.

In one embodiment, the DBM is in an amount of about 28 wt. % based onthe total weight of the injectable implant, the alginate is in an amountof about 6 wt. % based on the total weight of the injectable implant,and the phosphate buffered saline is in an amount of about 66 wt. %based on the total weight of the injectable implant.

The injectable implant further comprises sterile water. In someembodiments, the sterile water is in an amount of about 1 to about 50wt. % based on the total weight of the injectable implant. In someembodiments, the sterile water is in an amount of from about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, to about 50 wt. % based on the totalweight of the injectable implant.

In various embodiments, the injectable implant has an inherent viscosity(IV) of from about 0.1 dL/g to about 1.2 dL/g or from about 0.20 dL/g toabout 0.50 dL/g. Other IV ranges include but are not limited to about0.05 to about 0.15 dL/g, about 0.10 to about 0.20 dL/g, about 0.15 toabout 0.25 dL/g, about 0.20 to about 0,30 dL/g, about 0.25 to about 0.35dL/g, about 0.30 to about 0.35 dL/g, about 0.35 to about 0.45 dL/g,about 0.40 to about 0.45 dL/g, about 0.45 to about 0.55 dL/g, about 0.50to about 0.70 dL/g, about 0.55 to about 0.6 dL/g, about 0.60 to about0.80 dL/g, about 0.70 to about 0.90 dL/g, about 0.80 to about 1.00 dL/g,about 0.90 to about 1.10 dL/g, about 1.0 to about 1.2 dL/g, about 1.1 toabout 1.3 dL/g, about 1.2 to about 1.4 dL/g, about 1.3 to about 1,5dL/g, about 1.4 to about 1.6 dL/g, about 1.5 to about 1.7 dL/g, about1.6 to about 1.8 dL/g, about 1.7 to about 1.9 dL/g, or about 1.8 toabout 2.1 dL/g.

In some embodiments, the injectable implant may have a pre-dosedviscosity in the range of about 1 to about 3000 centipoise (cps), 1 toabout 2000 cps, 1 to about 1500 cps, 1 to about 1000 cps. 1 to about 500cps, 1 to about 300 cps, or 1 to about 100 cps. In some embodiments, theinjectable implant may have a pre-dosed viscosity of from about 1, 50,100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750,800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350,1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950,2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550,2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, to about 3000 cps.

In some embodiments, a viscosity enhancing agent is added to theinjectable implant in an amount from about 0.1 to about 20 wt. % of thetotal weight of the injectable implant. In some embodiments, a viscosityenhancing agent is added to the injectable implant in an amount of fromabout 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0,9, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 to about 20 wt. % of thetotal weight of the injectable implant. In some embodiments, theviscosity enhancing agent comprises mannitol, trehalose, hydroxypropylcellulose, hydroxypropyl methylcellulose, hydroxyethyl methylcellulose,carboxymethylcellulose and salts thereof, Carbopol,poly-(hydroxyethyl-methacrylate), poly-(methoxyethylmethacrylate),poly(methoxyethoxyethylmethacrylate), polymethyl-methacrylate (PMMA),methylmethacrylate (MMA), gelatin, polyvinyl alcohols, propylene glycol,mPEG, PEG 200, PEG 300, PEG 400, PEG 500, PEG 600, PEG 700, PEG 800, PEG900, PEG 1000, PEG 1450, PEG 3350, PEG 4500, PEG 8000 or combinationsthereof.

In some embodiments, the injectable implant has a modulus of elasticityin the range of 150 to about 2200 Pascals (Pa). In some embodiments, theinjectable implant has a modulus of elasticity in the range of 150 toabout 300, from about 150 to about 500, from about 150 to about 800.from about 150 to about 1000, from about 150 to about 1300, from about150 to about 1500, from about 150 to about 1800, from about 150 to about2000, from about 500 to about 1000, from about 500 to about 1300, fromabout 500 to about 1500, from about 500 to about 1800, from about 500 toabout 2000, from about 500 to about 2200, from about 1000 to about 1300,from about 1000 to about 1500, from about 1000 to about 1800, from about1000 to about 2000, from about 1000 to about 2200, from about 1300 toabout 1500, from about 1300 to about 1800, from about 1300 to about2000, from about 1300 to about 2200, from about 1500 to about 1800, fromabout 1500 to about 2000, from about 1500 to about 2200, from about 1800to about 2000, from about 1800 to about 2200, from about 2000 to about2200 Pa.

In sonic embodiments, the injectable implant has a density of betweenabout 2 g/cm³, and about 0.01 g/cm³. In some embodiments, the injectableimplant has a density of between about 1.5 g/cm³, and about 0.05 g/cm³.For example, the density may be less than about 2 g/cm³, 1.5 g/cm³, 1g/cm³, less than about 0.7 g/cm³, less than about 0.6 g/cm³, less thanabout 0.5 g/cm³, less than about 0.4 g/cm³, less than about 0.3 g/cm³,less than about 0.2 g/cm³, or less than about 0.1 g/cm³.

In some embodiments, if the injectable implant is to be placed in thespinal area, then the injectable implant can comprise preservative freematerial.

In some embodiments, a moldable implant 100 is provided that isconfigured to fit at or near a bone defect to promote bone growth, asshown in FIG. 2. In some embodiments, the bone defect is a bone void andthe moldable implant is configured to be a bone void filler. In someembodiments, the moldable implant is a putty. The moldable implant isconfigured to be moldable to any desired shape to fit the bone defectsite. The shape of the moldable implant may be tailored to the site atwhich it is to be situated.

In some embodiments, a medical practitioner may mold the moldableimplant into a desired shape and allow the moldable implant to cure ordry prior to implantation. In some embodiments, the moldable implant ismalleable in vivo. In such embodiments, a medical practitioner may moldthe implant directly into a bone defect site. The moldable implant ismalleable and configured to be pressed into a bone defect site to fillout crevices in a bone defect site. In some embodiments, the moldableimplant is malleable when wetted and is configured to remain malleablewhile in contact with the bone defect site. In some embodiments, themoldable implant can be formed to fit into the void space of aninterbody cage or around the outside of the cage in the intervertebralspace.

The moldable implant comprises lyophilized DBM 120 being in fiber,particle and chip forms, and an alginate 140. In the embodiment shown,the DBM fibers, DBM particles and DBM chips are uniformly distributedthroughout the alginate. The alginate holds the DBM fibers, DBMparticles and DBM chips together to form a cohesive moldable implant.The moldable implant also does not contain collagen so that animmunogenic reaction does not occur in patients that are administeredthe moldable implant.

The moldable implant is aseptically processed to enhanceosteoinductivity and osteoconductivity of bone. Aseptically processingtechniques as described below, prevent natural properties of bone frombeing striped, thus increasing the production of new bone formation viaosteoconduction and osteoinduction.

In some embodiments, the DBM is fully demineralized. In someembodiments, the fully demineralized DBM contains about less than 8, 7,6, 5, 4, 3, 2 and/or 1% of its original mineral content. In someembodiments, the DBM is partially demineralized, surface demineralizedand/or fully demineralized as mentioned above.

In some embodiments, the DBM is in fiber, particle and chip forms. Insome embodiments, the DBM fibers have a size of from about 1 to about 7mm. In some embodiments, the fibers have a size from about 1 to about 20mm, from about 1 to about 15 mm, from about 1 to about 10 mm, from about2 to about 15 mm, from about 2 to about 10 mm, from about 2 to about 5mm, from about 3 to about 1.5 mm, from about 3 to about 10 mm, fromabout 1 to about 5 mm, from about 4 to about 15 mm, from about 10 mm,from about 4 to about 5 mm, from about 5 to about 15 mm, or from about 5to about 10 mm. In some embodiments, the fibers have a size from about 1to about 50 mm, from about 5 to about 40 mm, from about 5 to about 30mm, from about 5 to about 10 mm. In some embodiments, the fibers have asize of from about 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 to about 50mm.

In some embodiments, the DBM particles have a size of from about 100microns to about 1000 microns. In some embodiments, the particles have asize of from about 100 microns to about 500 microns, from about 100microns to about 250 microns, from about 200 microns to about 1000microns, from about 200 microns to about 800 microns, from about 200microns to about 600 microns, from about 200 microns to about 400microns, from about 200 microns to about 300 microns, from about 300microns to about 800 microns, from about 300 microns to about 600microns, from about 300 microns to about 400 microns, from about 400microns to about 1000 microns, from about 400 microns to about 600microns, from about 500 microns to about 1000 microns, from about 500microns to about 800 microns, from about 500 microns to about 600microns.

In some embodiments, the DBM particles have a size from about 1, 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170,175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240,245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310,315, 320, 325, 330, 335, 340, 345. 350, 355, 360, 365, 370, 375, 380,385, 390, 395, 400, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445,450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515,520, 525 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585,590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655,660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725,730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795,800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865,870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935,940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995 to about 1000microns,

In some embodiments, the DBM chips have a size range of from about 1 toabout 4 mm. In some embodiments, the chips have a size range of fromabout 0.1 to about 50 mm, from about 0.1 to about 30 mm, from about 0.5to about 20 mm, from about 1 to about 40 mm, from about 1 to about 30mm, from about 1 to about 20 mm, from about 1 to about 15 mm, from about1 to about 10 mm, from about 1 to about 5 mm, from about 1 to about 3mm, from about 1 to about 2 mm, from about 2 to about 15 mm, from about2 to about 10 mm, from about 2 to about 5 mm, from about 3 to about 15mm, from about 3 to about 10 mm, from about 4 to about 15 mm, from about4 to about 5 mm, from about 5 to about 15 mm, or from about 5 to about10 mm.

In some embodiments, the DBM chips have a size of from about 0.1, 0.2.0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 44, 45, 46 47 48,49 to about 50 mm.

In some embodiments, the DBM is in an amount of about 15 wt. % to about40 wt. % based on a total weight of the moldable implant. In someembodiments, the DBM is in an amount of from about 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, to about 40 wt. % of the total weight of the implant. In someembodiments, the DBM is in an amount of about 1 to about 99 wt. % of thetotal weight of the implant. In some embodiments, the DBM is in anamount of from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36 37 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 8788 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, to about 99 wt. % of thetotal weight of the implant.

In some embodiments, the moldable implant comprises a ratio of fiber,particle and chip forms of about 33:33:33. In some embodiments, themoldable implant comprises a ratio of fiber, particle and chip forms offrom about 1:2:1, 2:1:1, 1:2:2, 1:3:1, 3:1:1, 1:3:3, 1:4:1, 4:1:1,1:4:4, 1:5:1, 5:1:1, 1:5:5, 1:6:1, 6:1:1, 1:1:6, 1:7:1, 7:1:1, 1:1:7,1:8:1, 8:1:1, 1:1:8, 1:9:1, 9:1:1, 1:1:9, 1:10:1, 10:1:1 to about1:1:10.

In some embodiments, from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 to about 99% ofthe DBM comprises the fiber forty e. fibers). In some embodiments, fromabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98 to about 99% of the DBM comprises theparticle form (e.g., particles). In some embodiments, about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 7 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, to about 99% of the DBM comprises the chip form (e.g.,chips).

The alginate holds the DBM particles, DBM fiber, and/or the DBM chipstogether in the injectable putty or moldable implant. In someembodiments, the alginate is in an amount of from about 3 wt. % to about20 wt. % based on the total weight of the moldable implant. In someembodiments, the alginate is in an amount of from about 5 to about 15wt. %, of from about 10 to about 20 wt. % or of from about 1 to 5 wt. %of the total weight of the moldable implant. In some embodiments, thealginate is in an amount of from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36 37 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87 88 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, to about99 wt. % of the total weight of the implant.

In some embodiments, the alginate is a sterile sodium alginate. In someembodiments, the alginate is sodium alginate, potassium alginate,calcium alginate, ammonium alginate, propylene glycol alginate, or acombination thereof.

In some embodiments, the alginate is in a powder form and has a particlesize of from about 100 to about 1,000 microns. In some embodiments, theparticle size of the alginate is of from about 100 microns to about 500microns, from about 100 microns to about 250 microns, from about 200microns to about 1000 microns, from about 200 microns to about 800microns, from about 200 microns to about 600 microns, from about 200microns to about 400 microns, from about 200 microns to about 300microns, from about 300 microns to about 800 microns, from about 300microns to about 600 microns, from about 300 microns to about 400microns, from about 400 microns to about 1000 microns, from about 400microns to about 600 microns, from about 500 microns to about 1000microns, from about 500 microns to about 800 microns, from about 500microns to about 600 microns.

In some embodiments, the alginate powder has a particle size of fromabout 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155,160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225,230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295,300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365,370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435,440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505,510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575,580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645,650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715,720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785,790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855,860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925,930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995 toabout 1000 microns.

In some embodiments, the alginate has an average molecular weight ofabout 100,000 to about 600,000 Daltons (Da). In some embodiments, thealginate has an average molecular weight of from about 100,000 to about500,000 Da, or of from about 200,000 to about 400,000 Da. In someembodiments, the alginate has an average molecular weight of from about200,000, 225,000, 250,000, 275,000, 300,000, 325,000, 350,000, 375,000,400,000, 425,000, 450,000, 475,000, 500,000 525,000, 550,000, 575,000 toabout 600,000 Da.

In some embodiments, when the alginate powder is mixed in an aqueouscarrier, such as phosphate buffered saline, the alginate comprises aviscosity of about 20 to about 2,000 centipoises (cps). In someembodiments, the alginate comprises a viscosity of from about 20, 30,40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320,330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460,470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600,610, 620, 630, 640, 650 660, 670, 680, 690, 700, 710, 720, 730, 740,750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880,890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020,1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140,1150, 1160, 1170, 1180, 1190, 1200, 1210, 1220, 1230, 1240, 1250, 1260,1270, 1280, 1290, 1300, 1310, 1320, 1330, 1340, 1350, 1360, 1370, 1380,1390, 1400, 1410, 1420, 1430, 1440, 1450, 1460, 1470, 1480, 1490, 1500,1510, 1520, 1530, 1540, 1550, 1560, 1570, 1580, 1590, 1600, 1610, 1620,1630, 1640, 1650, 1660, 1670, 1680, 1690, 1700, 1710, 1720, 1730, 1740,1750, 1760, 1770, 1780, 1790, 1800, 1810, 1820, 1830, 1840, 1850, 1860,1870, 1880, 1890, 1900, 1910, 1920, 1930, 1940, 1950, 1960, 1970, 1980,1990 to about 2000 cps.

The moldable implant further comprises an aqueous carrier comprisingphosphate buffered saline 160. The aqueous carrier is uniformlydistributed throughout the implant and is used to allow a uniformlymixed implant.

The phosphate buffered saline is in an amount from about 40 wt. % toabout 60 wt. % based on the total weight of the moldable implant. Insome embodiments, the phosphate buffered saline is in an amount of fromabout 40 wt. % to about 50 wt. % or from about 50 wt. % to about 60 wt,% of the total weight of the moldable implant. In some embodiments, thephosphate buffered saline is in an amount of from about 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, to about60 wt. % of the total weight of the moldable implant.

In one embodiment, the DBM is in an amount of about 30 wt. % based onthe total weight of the moldable implant, the alginate is in an amountof about 14 wt. % based on the total weight of the moldable implant, andthe phosphate buffered saline is in an amount of about 54 wt. % based onthe total weight of the moldable implant.

The moldable implant further comprises sterile water. In someembodiments, the sterile water is in an amount of about 1 to about 50wt. % based on the total weight of the moldable implant. In someembodiments, the sterile water is in an amount of about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, to about 50 wt. % based on the total weightof the moldable implant.

In various embodiments, the moldable implant has an inherent viscosity(IV) of from about 0.10 dL/g to about 1.2 dL/g or from about 0.20 dL/gto about 0.50 dL/g. Other IV ranges include but are not limited to about0.05 to about 0.15 dL/g, about 0.10 to about 0.20 dL/g, about 0.15 toabout 0.25 dL/g, about 0.20 to about 0.30 dL/g, about 0.25 to about 0.35dL/g, about 0.30 to about 0.35 dL/g, about 0.35 to about 0.45 dL/g,about 0.40 to about 0.45 dL/g, about 0.45 to about 0.55 dL/g, about 0.50to about 0.70 dL/g, about 0.55 to about 0.6 dL/g, about 0.60 to about0.80 dL/g, about 0,70 to about 0.90 dL/g, about 0.80 to about 1.00 dL/g,about 0.90 to about 1.10 dL/g, about 1.0 to about 1.2 dL/g, about 1.1 toabout 1.3 dL/g, about 1.2 to about 1.4 dL/g, about 1.3 to about 1.5dL/g, about 1.4 to about 1.6 dL/g, about 1.5 to about 1.7 dL/g, about1.6 to about 1.8 dL/g, about 1.7 to about 1.9 dL/g, or about 1.8 toabout 2.1 dL/g.

In some embodiments, a viscosity enhancing agent is added to themoldable implant in an amount from about 0.1 to about 20 wt. % of thetotal weight of the moldable implant. In some embodiments, a viscosityenhancing agent is added to the moldable implant in an amount of about0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17 18, 19 or about 20 wt. % of the totalweight of the moldable implant. In some embodiments, the viscosityenhancing agent comprises mannitol, trehalose, hydroxypropyl cellulose,hydroxypropyl methylcellulose, hydroxyethyl methylcellulose,carboxymethylcellulose and salts thereof, Carbopol,poly-(hydroxyethyl-methacrylate), poly-(methoxyethylmethacrylate),poly(methoxyethoxyethylmethacrylate), polymethyl-methacrylate (PMMA),methylmethacrylate (MMA), gelatin, polyvinyl alcohols, propylene glycol,mPEG, PEG 200, PEG 300, PEG 400, PEG 500, PEG 600, PEG 700, PEG 800, PEG900, PEG 1000, PEG 1450, PEG 3350, PEG 4500, PEG 8000 or combinationsthereof.

In some embodiments, the moldable implant has a modulus of elasticity inthe range of 150 to about 2200 Pascals (Pa). In some embodiments, themoldable implant has a modulus of elasticity in the range of 150 toabout 300, from about 150 to about 500, from about 150 to about 800,from about 150 to about 1000, from about 150 to about 1300, from about150 to about 1500, from about 150 to about 1800, from about 150 to about2000, from about 500 to about 1000, from about 500 to about 1300, fromabout 500 to about 1500, from about 500 to about 1800, from about 500 toabout 2000, from about 500 to about 2200, from about 1000 to about 1300,from about 1000 to about 1500, from about 1000 to about 1800, from about1000 to about 2000, from about 1000 to about 2200, from about 1300 toabout 1500, from about 1300 to about 1800, from about 1300 to about2000, from about 1300 to about 2200, from about 1500 to about 1800, fromabout 1500 to about 2000, from about 1500 to about 2200, from about 1800to about 2000, from about 1800 to about 2200, from about 2000 to about2200 Pa.

In some embodiments, the moldable implant has a density of between about2 g/cm³, and about 0.01 g/cm³. In some embodiments, the moldable implanthas a density of between about 1.5 g/cm³, and about 0.05 g/cm³. Forexample, the density may be less than about 2 g/cm³, 1.5 g/cm³, 1 g/cm³,less than about 0.7 g/cm³, less than about 0.6 g/cm³, less than about0.5 g/cm³, less than about 0.4 g/cm³, less than about 0.3 g/cm³, lessthan about 0.2 g/cm³, or less than about 0.1 g/cm³.

In some embodiments, if the moldable implant is to be placed in thespinal area, then the moldable implant will comprise preservative freematerial.

In some embodiments, the aqueous carrier comprising phosphate bufferedsaline can alternatively or further include a variety of additionalfluids. In some embodiments, the aqueous carrier comprises phosphatebuffered saline, sterile water, physiological saline, sodium chloride,dextrose, Lactated Ringer's solution, or a combination thereof.

In some embodiments, the injectable implant and/or the moldable implantfurther comprises hyaluronic acid, cellulose ethers (such ascarboxymethylcellulose), collagen, gelatin, autoclaved bone powder,osteoconductive carriers, whole blood, blood fractions, bone marrowaspirate, concentrated bone marrow aspirate, and mixtures thereof.Non-limiting examples of blood fractions include serum, plasma,platelet-rich plasma, concentrated platelet-rich plasma, platelet-poorplasma, and concentrated platelet poor plasma.

In some embodiments, the injectable and/or moldable implant is porous toallow influx of at least bone and/or cartilage cells therein. By“porous,” it is meant that the implant has a plurality of pores. Thepores of the implant are a size large enough to allow influx of blood,other bodily fluid, and progenitor and/or bone and/or cartilage cellsinto the interior to guide the process of tissue formation in vivo inthree dimensions.

In some embodiments, the injectable and/or moldable implant comprisespores having a pore size from about 1 micron to about 500 microns. Insome embodiments, the pore size is from about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12., 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300,310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440,440, 450, 460, 470, 480, 490 to about 500 microns.

In some embodiments, the injectable and/or moldable implant has aporosity of at least about 30%, at least about 50%, at least about 60%,at least about 70%, at least about 90% or at least about 95%, or atleast about 99%. The pores may support ingrowth of cells, formation orremodeling of bone, cartilage and/or vascular tissue.

In some embodiments, the moldable implant can be molded by the surgeonto the desired shape to fit the tissue or bone defect. In someembodiments, the moldable implant has a size of from about 5 mm to about500 mm. In some embodiments, the moldable implant has a size of aboutfrom about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 2, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 110,120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250,260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390,400, 410, 420, 430, 440, 440, 450, 460, 470, 480, 490 to about 500 mm.

Additional Biodegradable Polymers

In some embodiments, the injectable and/or moldable implant in additionto alginate or as an alternative to alginate comprises one or morebiodegradable polymers. In some embodiments, the biodegradable polymeris crosslinked. In an alternative embodiment, the injectable and/ormoldable implant comprises human collagen, as well as recombinantcollagen or combinations thereof. Examples of suitable collagen include,but are not limited to, human collagen type I, human collagen type IIhuman collagen type III, human collagen type IV, human collagen type V,human collagen type VI, human collagen type VII, human collagen typeVIII, human collagen type IX, human collagen type X, human collagen typeXI, human collagen type XII, human collagen type XIII, human collagentype XIV, human collagen type XV, human collagen type XVI, humancollagen type XVII, human collagen type XVIII, human collagen type XIX,human collage type XX, human collagen type XXI, human collagen typeXXII, human collagen type XXIII, human collagen type XXIV, humancollagen type XXV, human collagen type XXVI, human collagen type XXVII,and human collagen type XXVIII, or combinations thereof. Collagenfurther may comprise hetero- and homo-trimers of any of theabove-recited collagen types. In some embodiments, the collagencomprises hetero- or homo-trimers of human collagen type I, humancollagen type II, human collagen type III; or combinations thereof. Invarious embodiments, the collagen may be crosslinked.

In some alternate embodiments, the injectable and/or moldable implantcomprises collagen-containing biomaterials from the implant marketwhich, when placed in a bone defect, provide scaffolding around whichthe patient's new bone and/or cartilage will grow, gradually replacingthe injectable and/or moldable implant as the target site heals.Examples of suitable carrier matrices may include, but are not limitedto, the MasterGraft® Matrix produced by Medtronic Sofamor Danek, Inc.,Memphis, Tenn.; MasterGraft® Putty produced by Medtronic Sofamor Danek,Inc., Memphis, Tenn.; Absorbable Collagen Sponge (“ACS”) produced byIntegra LifeSciences Corporation, Plainsboro, N.J.; bovine skin collagenfibers coated with hydroxyapatite, e.g., Healos®, marketed by Johnson &Johnson, USA; collagen sponges, e.g. Hemostagene® marketed by Coletica SA, France, or e.g., Helisat® marketed by Integra Life Sciences Inc.,USA; and Collagraft® Bone Graft Matrix produced by Zimmer Holdings,Inc., Warsaw, Ind.

In some alternate embodiments, the collagen contains both solublecollagen and insoluble collagen fibers. The soluble collagen andinsoluble collagen fibers can first be prepared separately, and thencombined. Both the soluble collagen and the insoluble collagen fibersare derived from human sources.

In some embodiments, the injectable and/or moldable implant in additionto alginate or as an alternative to alginate comprises a biodegradablepolymeric or non-polymeric material. In some embodiments, the injectableand/or moldable implant may include a biodegradable polymer. Forexample, the biodegradable polymer can comprise polyether ether ketone(PEEK). In some embodiments, the injectable and/or moldable implant maycomprise one or more of poly (alpha-hydroxy acids), polyglycolide (PG),polyethylene glycol (PEG) conjugates of poly (alpha-hydroxy acids),polyorthoesters (POE), polyaspirins, polyphosphagenes, gelatin,hydrolyzed gelatin, fractions of hydrolyzed gelatin, elastin, starch,pre-gelatinized starch, hyaluronic acid, chitosan, alginate, albumin,fibrin, vitamin E analogs, such as alpha tocopheryl acetate, d-alphatocopheryl succinate, D,L-lactide, or L-lactide, caprolactone, dextrans,vinylpyrrolidone, polyvinyl alcohol (PVA), PVA-g-PLGA, PEGT-PBTcopolymer (polyactive), methacrylates, PEO-PPO-PAA copolymers,PLGA-PEO-PLGA, PEG-PLG, PLA-PLGA, poloxamer 407, PEG-PLGA-PEG triblockcopolymers, POE, SAIB (sucrose acetate isobutyrate), polydioxanone,methylmethacrylate (MMA), MMA and N-vinylpyyrolidone, polyamide,oxycellulose, copolymer of glycolic acid and trimethylene carbonate,polyesteramides, polyether ether ketone, polymethylmethacrylate,silicone, hyaluronic acid, or combinations thereof.

In some embodiments, the injectable and/or moldable implantalternatively or in addition comprises at least one biodegradablepolymer comprising one or more of poly(lactide-co-glycolide) (PLGA),polylactide (PLA), polyglycolide (PGA), D-lactide, D,L-lactide,L-lactide, D,L-lactide-co-ε-caprolactone, L-lactide-co-εcaprolactone,D,L-lactide-co-glycolide-co-ε-caprolactone,poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone),poly(D-lactide-co-caprolactone), poly(D,L-lactide), poly(D-lactide),poly(L-lactide), poly(esteramide) or a combination thereof.

In some embodiments, the injectable and/or moldable implantalternatively or in addition to alginate comprises one or more polymers(e.g., PLA, PLGA, etc.) having a MW of from about 15,000 to about150,000 Da or from about 25,000 to about 100,000 Da.

DBM/Additional Particles

In alternative embodiments, the implant comprises mineral particles,such as, for example, ceramics. In some embodiments, the particles inthe implant comprise a resorbable ceramic, bone, synthetic degradablepolymer, hyaluronic acid, chitosan or combinations thereof. In someembodiments, the particles comprise cortical, cancellous, and/orcorticocancellous, allogenic, xenogeneic or transgenic bone tissue. Thebone component can comprise, consist essentially of or consist of fullymineralized, partially demineralized, fully demineralized orcombinations thereof. In some embodiments, the mineral particlescomprise, consist essentially of or consist of bone powder,demineralized bone powder, porous calcium phosphate ceramics,hydroxyapatite, tricalcium phosphate, bioactive glass or combinationsthereof.

In some alternative embodiments, the implant may comprise a resorbableceramic (e.g., hydroxyapatite, tricalcium phosphate, bioglasses, calciumsulfate, etc.) tyrosine-derived polycarbonate poly (DTE-co-DTcarbonate), in which the pendant group via the tyrosine an amino acid iseither an ethyl ester (DTE) or free carboxylate (DT) or combinationsthereof.

In some alternative embodiments, the implant may contain an inorganicmaterial, such as an inorganic ceramic and/or bone substitute material.Exemplary inorganic materials or bone substitute materials include butare not limited to aragonite, dahlite, calcite, brushite, amorphouscalcium carbonate, vaterite, weddellite, whewellite, struvite, urate,ferrihydrate, francolite, monohydrocalcite, magnetite, goethite, dentin,calcium carbonate, calcium sulfate, calcium phosphosilicate, sodiumphosphate, calcium aluminate, calcium phosphate, hydroxyapatite,alpha-tricalcium phosphate, dicalcium phosphate, β-tricalcium phosphate,tetracalcium phosphate, amorphous calcium phosphate, octacalciumphosphate, BIOGLASS™ fluoroapatite, chlorapatite, magnesium-substitutedtricalciwn phosphate, carbonate hydroxyapatite, substituted forms ofhydroxyapatite (e.g., hydroxyapatite derived from bone may besubstituted with other ions such as fluoride, chloride, magnesiumsodium, potassium, etc.), or combinations or derivatives thereof.

In some embodiments, by including inorganic ceramics, such as forexample, calcium phosphate, in the implant, this will act as a localsource of calcium and phosphate to the cells attempting to deposit newbone. The inorganic ceramic also provides compression resistance andload bearing characteristics to the implant.

In some embodiments, the mineral particles in the implant comprisetricalcium phosphate and hydroxyapatite in a ratio of about 80:20 toabout 90:10. In some embodiments, the mineral particles in the implantcomprise tricalcium phosphate and hydroxyapatite in a ratio of about70:30 to about 95:5. In some embodiments, the mineral particles in theimplant comprise tricalcium phosphate and hydroxyapatite in a ratio ofabout 85:15.

In some embodiments, as discussed above, the implant containsdemineralized bone material disposed therein. In sonic embodiments, thedemineralized bone material can comprise demineralized bone, particles,powder, chips, triangular prisms, spheres, cubes, cylinders, shards,fibers or other shapes having irregular or random geometries. These caninclude, for example, “substantially demineralized,” “partiallydemineralized,” or “fully demineralized” cortical and cancellous bone.These also include surface demineralization, where the surface of thebone construct is substantially demineralized, partially demineralized,or fully demineralized, yet the body of the bone construct is fullymineralized. In some embodiments, the configuration of the bone materialcan be obtained by milling, shaving, cutting or machining whole bone.

In some alternative embodiments, the implant comprises elongateddemineralized bone fibers having an average length to average thicknessratio or aspect ratio of the fibers from about 50:1 to about 1000:1. Inoverall appearance, the elongated demineralized bone fibers can be inthe form of threads, narrow strips, or thin sheets. The elongateddemineralized bone fibers can be substantially linear in appearance orthey can be coiled to resemble springs. In some embodiments, theelongated demineralized bone fibers are of irregular shapes including,for example, linear, serpentine or curved shapes. The elongated bonefibers can be demineralized however some of the original mineral contentmay be retained when desirable for a particular embodiment.

Expandable Phase

In some embodiments, the implant may comprise a material, such as, forexample, an expandable phase, to facilitate swelling of the implant. Theexpandable phase comprises polymers that swell upon taking in fluid(e.g., saline, water, bodily fluid, etc.), and thus increase the volumeof the implant and which further holds the implant in position overtime.

In some embodiments, the expandable phase comprises a range of fromabout 0.1% to about 20% based on the total weight of the implant. Insome embodiments, the expandable phase comprises a range of from about0.1% to about 10% based on the total weight of the implant. In sonicembodiments, the expandable phase comprises from about 0.1%, 0.25%,0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or to about 10% based onthe total weight of the implant.

In some embodiments, the expandable phase comprises polymers, monomers,starches, gums, poly(amino acids) or a combination thereof that swellupon contact with fluid (water, body fluids, etc.). in variousembodiments, the amount of swelling can range from about 5 to about 100percent, from about 5 to about 40 percent, or from about 5 to about 20percent. The time to reach maximum swelling can be varied depending onthe location and desired property of the implant. In practice, the timeto reach maximum swelling can occur within a period of 5 days, 3 days, 2days or within a period of 24 hours.

A suitable swellable material may include, for example, hydroxypropylcellulose, hydroxypropyl methylcellulose, hydroxyethyl methylcellulose,carboxymethylcellulose, hydroxyethylcellulose and salts thereof,Carbopol, poly(hydroxyethylmethacrylate), poly(methoxyethylmethacrylate)poly(methoxyethoxy-ethylmethacrylate), polymethylmethacrylate (PMMA),methylmethacrylate (MMA), gelatin, polyvinyl alcohols, propylene glycol,PEG 200, PEG 300, PEG 400, PEG 500, PEG 550, PEG 600, PEG 700, PEG 800PEG 900, PEG 1000, PEG 1450, PEG 3350, PEG 4500, PEG 8000 orcombinations thereof. In some embodiments, the expandable phase includesgelling polymers including but not limited to cellulosic polymers, vinylpolymers, such as polyvinylpyrrolidone; acrylic polymers and copolymers,such as acrylic acid polymer, methacrylic acid copolymers, ethylacrylate-methyl methacrylate copolymers, or the like; or mixturesthereof.

A non-limiting list of swellable materials which the expandable phasemay comprise include polyvinyl alcohol (PVA), PVA modified withhydrophilic co-monomers, e.g., AMPS, PVA modified with fast crosslinkinggroups, e.g., NAAADA, PVA modified with polyvinylpyrroline (PVP),carboxymethylcellulose, polyethylene glycol (PEG), poly(vinyl ether),co-polymers of PVA and PEG, polypropylene glycol (PPG), co-polymers ofPEG and PPG, co-polymers of PVA or PPG, polyacrylonitrile,hydrocolloids, e.g. agar, alginates, collagen, elastin, chitin,chitosan, gelatin, sugar, mannitol, or the like. In various embodiments,the swellable material includes, for example,poly(N-isopropylacrylamide-co-acrylic acid)-poly(L-lactic acid) (NAL);poly(N-isopropyl acrylamide) (PNIPAM) grafted to other polymers such ascarboxymethylcellulose (CMC) copolymers or polymers including blockcopolymers and end-functionalized polymers, composites or copolymerscontaining thermo-sensitive poly(2-ethoxyethyl vinyl ether) and/orpoly(hydroxyethyl vinyl ether) and/or (EOVE200-HOVE400), whose sol-geltransition temperature is 20.5° C.

In some embodiments, the expandable phase includes hyaluronic acid. Insome embodiments, the expandable phase includes glycosaminoglycans.Non-limiting examples of glycosaminoglycans include chondroitin sulfate,dermatan sulfate, keratin sulfate, heparin, heparin sulfate, andhyaluronan. In some embodiments, the expandable phase includes mannitol,PEG, magnesium alginate or glycerol.

The swellable polymers may be crosslinked or lightly crosslinkedhydrophilic polymers. Although these polymers may be non-ionic,cationic, zwitterionic, or anionic, in various embodiments, theswellable polymers are cationic or anionic. In various embodiments, theswellable polymer may contain a multiplicity of acid functional groups,such as carboxylic acid groups, or salts thereof. Examples of suchpolymers suitable for use herein include those which are prepared frompolymerizable, acid-containing monomers, or monomers containingfunctional groups which can be converted to acid groups afterpolymerization. Examples of such polymers also includepolysaccharide-based polymers such as carboxymethyl starch andcellulose, and poly(amino acid) polymers such as poly(aspartic acid).Some non-acid monomers may also be included, usually in minor amounts,in preparing the absorbent polymers. Such non-acid. monomers include,for example, monomers containing the following types of functionalgroups: carboxylate or sulfonate esters, hydroxyl groups, amide groups,amino groups, nitrile groups, quaternary ammonium salt groups, and arylgroups (e.g. phenyl groups, such as those derived from styrene monomer).Other potential non-acid monomers include unsaturated hydrocarbons suchas ethylene, propylene, 1-butene, butadiene, or isoprene.

In some embodiments, the expandable phase comprises substances which arecapable of becoming freely permeable following hydration in aqueousfluids. Such substances include polysaccharides, such as gelatin,saccharose, sorbitol, mannanes, jaturonic acid, polyaminoacids,polyalcohols, polyglycols, or the like. In addition to the foregoing,the swellable polymer may also include additional excipients such aslubricants, flow promoting agents, plasticizers, and anti-stickingagents. For example, the expandable phase may further includepolyethylene glycol, polyvinylpyrrolidone, talc, magnesium stearate,glyceryl behenate, stearic acid, or titanium dioxide.

In various embodiments, the particle size distribution of the expandablephase material may be about 10 micrometers, 13 micrometers, 85micrometers, 100 micrometers, 151 micrometers, 200 micrometers and allsubranges therebetween. In some embodiments, at least 75% of theparticles have a size from about 10 micrometers to about 200micrometers. In some embodiments, at least 85% of the particles have asize from about 10 micrometers to about 200 micrometers. In someembodiments, at least 95% of the particles have a size from about 10micrometers to about 200 micrometers. In some embodiments, all of theparticles have a size from about 10 micrometers to about 200micrometers. In some embodiments, at least 75% of the particles have asize from about 20 micrometers to about 180 micrometers. In someembodiments, at least 85% of the particles have a size from about 20micrometers to about 180 micrometers. In some embodiments, at least 95%of the particles have a size from about 20 micrometers to about 180micrometers. In some embodiments, all of the particles have a size fromabout 20 micrometers to about 180 micrometers.

Lyophilization

In some embodiments, the DBM fibers, particles and chips arelyophilized. The lyophilization process typically includes sublimationof water from a frozen formulation under controlled conditions.Lyophilization can be carried out using standard equipment as used forlyophilization or vacuum drying. The cycle may be varied depending uponthe equipment and facilities used for the fill and finish.

Initially, in some embodiments, the DBM is placed in a lyophilizationchamber under a range of temperatures and then subjected to temperatureswell below the freezing point of DBM, generally for several hours. Afterfreezing is complete, the lyophilization chamber and the condenser areevacuated through vacuum pumps, the condenser surface having beenpreviously chilled by circulating refrigerant. The condenser will havebeen chilled below the freezing point of the DBM. Additionally,evacuation of the chamber should continue until a pressure of about 50mTorr to about 600 mTorr, preferably about 50 to about 150 mTorr isobtained.

The lyophilized DBM is then warmed under vacuum in the chamber andcondenser. This usually will be carried out by warming the shelveswithin the lyophilizer on which the lyophilized DBM rests during thelyophilization process at a pressure ranging from about 50 mTorr toabout 600 mTorr. The warming process will optimally take place verygradually, over the course of several hours. Complete drying can beaccomplished by stabilization of vacuum, condenser temperature andlyophilized DBM shelf temperature. After the initial drying, thetemperature of the lyophilized DBM can be increased and maintained forseveral hours. Once the drying cycle is completed, the pressure in thechamber can be slowly released to atmospheric pressure (or slightlybelow) with sterile, dry-nitrogen gas (or equivalent gas).

In some embodiments, after lyophilization, the DBM is from about 95 toabout 99.5% free of moisture. In various embodiments, the DBM is fromabout 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99, to about 99.5% free ofmoisture. In some embodiments, the DBM has about 0.5% to about 5%moisture content remaining after lyophilization. In various embodiments,the DBM has from about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 to about 5%moisture content remaining after lyophilization. Lyophilized DBM isstable and can be stored at a wide range of temperatures. LyophilizedDBM can be stored at or below 30° C., for example, refrigerated at 4°C., or at room temperature (e.g., approximately 25° C.).

Method of Making

In some embodiments, a method of making an injectable or moldableimplant is provided. The method comprises mixing lyophilizeddemineralized bone matrix (DBM) being in fiber and particle forms in anamount of about 28% based on a total weight of the implant, or DBM infiber, particle and chip forms in an amount of about 30% based on thetotal weight of the implant with an aqueous liquid comprising analginate so as to uniformly distribute the DBM within the alginate toform the injectable or moldable implant.

In some embodiments, the alginate is in an amount of about 1 % to about10 wt. % of the total weight of the implant or in an amount of about 3wt. % to about 20 wt. % of the total weight of the implant.

In some embodiments, the aqueous liquid comprises phosphate bufferedsaline. In some embodiments, the phosphate buffered saline is in anamount of about 50 wt. % to about 70 wt. % of the total weight of theimplant or in an amount of about 40 wt. % to about 60 wt. % of the totalweight of the implant.

In some embodiments, before the DBM is mixed with the aqueous liquid,the aqueous liquid is mixed with the alginate and formed into a gel, andthe gel is irradiated to sterilize the gel. In some embodiments, themixing step is performed aseptically. In some embodiments, sterilefiltered water is mixed concurrently with the DBM into the aqueousliquid. In some embodiments, the sterile filtered water is in an amountof from 1 to about 50 wt. % based on the total weight of the moldableimplant.

In some embodiments, the method further comprises loading the injectableimplant into a syringe after the implant is formed and inserting thesyringe into a pre-sterilized foil pouch. In some embodiments, themethod further comprises inserting the moldable implant into apre-sterilized foil pouch.

In some embodiments, before administration to a patient, in someinstances, the biodegradable polymer (e.g., alginate) can be subjectedto one or more additional operations such as heating, lyophilizingand/or crosslinking. In this regard, crosslinking can be used to improvethe strength of the implant. Crosslinking can be achieved, for example,by chemical reaction, the application of energy such as radiant energy(e.g., UV light or microwave energy), drying and/or heating anddye-mediated photo-oxidation; dehydrothermal treatment; enzymatictreatment or others.

In some embodiments, a chemical crosslinking agent is used. Examples ofsuitable cross-linking agents include those that contain bifunctional ormultifunctional reactive groups, and which react with the implant.Chemical crosslinking can be introduced by exposing the implant to achemical crosslinking agent, either by contacting it with a solution ofthe chemical crosslinking agent or by exposure to the vapors of thechemical crosslinking agent. The resulting material can then be washedto remove substantially all remaining amounts of the chemicalcrosslinker if needed or desired for the performance or acceptability ofthe final implant.

Suitable chemical crosslinking agents include mono- and dialdehydes,including glutaraldehyde and formaldehyde; polyepoxy compounds such asglycerol polyglycidyl ethers, polyethylene glycol diglycidyl ethers andother polyepoxy and diepoxy glycidyl ethers; tanning agents includingpolyvalent metallic oxides such as titanium dioxide, chromium dioxide,aluminum dioxide, zirconium salt, as well as organic tannins and otherphenolic oxides derived from plants; chemicals for esterification orcarboxyl groups followed by reaction with hydrazide to form activatedacyl azide functionalities in the collagen; dicyclohexyl carbodiimideand its derivatives as well as other heterobifunctional crosslinkingagents; hexamethylene diisocyante; and/or sugars, including glucose,will also crosslink the implant.

In some embodiments, radiographic markers can be included on the implantto permit the user to position it accurately into the target site of thepatient. These radiographic markers will also permit the user to trackmovement and degradation of the implant at the site over time. In thisembodiment_(;) the user may accurately position the implant in the siteusing any of the numerous diagnostic imaging procedures. Such diagnosticimaging procedures include, for example, X-ray imaging or fluoroscopy.Examples of such radiographic markers include, but are not limited to,ceramics, barium, phosphate, bismuth, iodine, tantalum, tungsten, and/ormetal beads or particles. In various embodiments, the radiographicmarker could be a spherical shape or a ring around the implant.

In some embodiments, the implant can be administered to the target siteby passing it through a “cannula” or “needle” that can be a part of adelivery device e.g., a syringe, a gun delivery device, or any medicaldevice suitable for the delivery of the implant to a targeted organ oranatomic region. The cannula or needle of the device is designed tocause minimal physical and psychological trauma to the patient.

The implant may be used to repair bone and/or cartilage at a targettissue site, e.g., one resulting from injury, defect brought aboutduring the course of surgery, infection, malignancy or developmentalmalformation. The implant can be utilized in a wide variety oforthopedic, periodontal, neurosurgical, oral and maxillofacial surgicalprocedures such as the repair of simple and/or compound fractures and/ornon-unions; external and/or internal fixations; joint reconstructionssuch as arthrodesis; general arthroplasty; cup arthroplasty of the hip;femoral and humeral head replacement; femoral head surface replacementand/or total joint replacement; repairs of the vertebral columnincluding spinal fusion and internal fixation; tumor surgery, e.g.,deficit filling; discectomy; laminectomy; excision of spinal cordtumors; anterior cervical and thoracic operations; repairs of spinalinjuries; scoliosis, lordosis and kyphosis treatments; intermaxillaryfixation of fractures; mentoplasty; temporomandibular joint replacement;alveolar ridge augmentation and reconstruction; inlay implantablematrices; implant placement and revision; sinus lifts; cosmeticprocedures; etc. Specific bones which can be repaired or replaced withthe implant herein include the ethmoid, frontal, nasal, occipital,parietal, temporal, mandible, maxilla, zygomatic, cervical vertebra,thoracic vertebra, lumbar vertebra, sacrum, rib, sternum, clavicle,scapula, humerus, radius, ulna, carpal bones, metacarpal bones,phalanges, ilium, ischium, pubis, femur, tibia, fibula, patella,calcaneus, tarsal and/or metatarsal bones.

Additional Therapeutic Agents

In some embodiments, the implant further comprises one or moreadditional therapeutic agents including one or more growth factors,statins, etc. Isolated osteoinductive agents that are included within animplant are typically sterile. In a non-limiting method, sterility isreadily accomplished for example by filtration through sterilefiltration membranes 0.2 micron membranes or filters). In oneembodiment, the implant includes osteoinductive agents comprising one ormore members of the family of Bone Morphogenetic Proteins (“BMPs”). BMPsare a class of proteins thought to have osteoinductive orgrowth-promoting activities on endogenous bone tissue, or function aspro-collagen precursors. Known members of the BMP family include, butare not limited to, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7,BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14 (GDF-5), BMP-15,BMP-16, BMP-17, BMP-18 as well as polynucleotides or polypeptidesthereof, as well as mature polypeptides or polynucleotides encoding thesame.

BMPs utilized as osteoinductive agents comprise one or more of BMP-1;BMP-2; BMP-3; BMP-4; BMP-5; BMP-6; BMP-7; BMP-8; BMP-9; BMP-10; BMP-11;BMP-12; BMP-13; BMP-15; BMP-16; BMP-17; or BMP-18; as well as anycombination of one or more of these BMPs, including full length BMPs orfragments thereof, or combinations thereof, either as polypeptides orpolynucleotides encoding the polypeptide fragments of all of the recitedBMPs. The isolated BMP osteoinductive agents may be administered aspolynucleotides, polypeptides, full length protein or combinationsthereof.

Indeed, the osteoinductive factors are the recombinant human bonemorphogenetic proteins (rhBMPs) because they are available in unlimitedsupply and do not transmit infectious diseases. In some embodiments, thebone morphogenetic protein is a rhBMP-2, rhBMP-4, rhBMP-7, orheterodimers thereof.

Recombinant BMP-2 can be used at a concentration of about 0.4 mg/mL, toabout 10.0 mg/mL, preferably near 1.5 mg/mL. However, any bonemorphogenetic protein is contemplated including bone morphogeneticproteins designated as BMP-1 through BMP-18. BMPs are available fromWyeth, Cambridge, Mass. and the BMPs and genes encoding them may also beprepared by one skilled in the art as described in U.S. Pat. No.5,187,076 to Wozney et al.; U.S. Pat. No. 5,366,875 to Wozney et al.;U.S. Pat. No. 4,877,864 to Wang et al.; U.S. Pat. No. 5,108,922 to Wanget al.; U.S. Pat. No. 5,116,738 to Wang et al.; U.S. Pat. No. 5,013,649to Wang et al,; U.S. Pat. No. 5,106,748 to Wozney et al.; and PCT PatentNos. WO93/00432 to Wozney et al.; WO94/26893 to Celeste et al.; andWO94/26892 to Celeste et al. All osteoinductive factors are contemplatedwhether obtained as above or isolated from bone. Methods for isolatingbone morphogenetic protein from bone are described, for example, in U.S.Pat. No. 4,294,753 to Urist and Urist et al., 81 PNAS 371, 1984.

In addition to the above, the implant may include one or more membersfrom the TGF-β superfamily. For example, the implant may include AMH,ARTN, GDF1, GDF10, GDF11, GDF15, GDE2, GDF3, GDF3A, GDF5, GDE6, GDF7,GDF8, GDF9, GDNF, INHA, INHBA, INHBB, INHBC, INHBE, LEFTY1, LEFTY2,MSTN, NODAL, NRTN, PSPN, TGFB1, TGFB2, TGFB3, FGF, basic FGF, VEGF,insulin-like growth factor, EGF, PDGF, nerve growth factor orcombinations thereof.

The growth factors of the present application may be disposed on or inthe implant with other therapeutic agents. For example, the growthfactor may be disposed on or in the implant by electrospraying,ionization spraying or impregnating, vibratory dispersion (includingsonication), nozzle spraying, compressed-air-assisted spraying, brushingand/or pouring.

Exemplary therapeutic agents include but are not limited to IL-1inhibitors, such Kineret® (anakinra), which is a recombinant,non-glycosylated form of the human interleukin-1 receptor antagonist(IL-1Ra), or AMG 108, which is a monoclonal antibody that blocks theaction of IL-1. Therapeutic agents also include excitatory amino acidssuch as glutamate and aspartate, antagonists or inhibitors of glutamatebinding to NMDA receptors, AMPA receptors, and/or kainate receptors.Interleukin-1 receptor antagonists, thalidomide (a TNF-α releaseinhibitor), thalidomide analogues (which reduce TNF-α production bymacrophages), quinapril (an inhibitor of angiotensin II, whichupregulates TNF-α), interferons such as IL-11 (which modulate TNF-αreceptor expression), and aurin-tricarboxylic acid (which inhibitsTNF-α), may also be useful as therapeutic agents for reducinginflammation. It is further contemplated that where desirable apegylated form of the above may be used. Examples of still othertherapeutic agents include NF kappa B inhibitors such as antioxidants,such as dithiocarbamate, and other compounds, such as, for example,sulfasalazine, statins or the like.

Examples of therapeutic agents suitable for use also include, but arenot limited to, an anti-inflammatory agent, analgesic agent, orosteoinductive growth factor or a combination thereof.

In some embodiments, additives are provided that can be disposed on orin the implant to enhance the DBM osteoinductive, osteoconductive,and/or osteopromotive activity. The additives include, but are notlimited to ascorbic acid in an amount of from about 0.001 wt. % to about3 wt. % based on the total weight of the implant, dexamethasone in anamount of from about 0.0001 wt. % to about 0.1 wt. % based on the totalweight of the implant, P-glycerophosphate in an amount of from about0.001 wt. % to about 3 wt. % based on the total weight of the implant,L-arginine in an amount of from about 0.001 wt. % to about 3 wt. % basedon the total weight of the implant, L-ascorbic acid-2-phosphate in anamount of from about 0.001 wt. % to about 3 wt. % based on the totalweight of the implant, nano-hydroxyapatite in an amount of from about0.1 wt. % to about 30 wt. % based on the total weight of the implant, ora combination thereof. The additive can also include zinc chloride in anamount of from about 0.001 wt. % to about 3 wt. % based on the totalweight of the implant.

Kits

The implant and/or the medical device to administer it may besterilizable. The implant is aseptically processed (e.g., not requiringterminal irradiation). in some embodiments, the implant may be packagedin a moisture resistant sterile package. in use, the surgeon removes oneor all components from the sterile package for use.

In various embodiments, a kit is provided comprising the implant. Thekit may include additional parts along with the implant combinedtogether to be used to administer the implant (e.g., wipes, needles,syringes, mixing syringe or other mixing device, etc.). The kit mayinclude the implant in a first compartment. The second compartment mayinclude a vial holding the carrier and any other instruments needed forthe delivery. A third compartment may include gloves, drapes, wounddressings and other procedural supplies for maintaining sterility of theimplanting process, as well as an instruction booklet, which may includea chart that shows how to administer the implant after reconstitutingit. A fourth compartment may include additional needles and/or sutures.Each tool may be separately packaged in a plastic pouch that issterilized. A fifth compartment may include an agent for radiographicimaging. A cover of the kit may include illustrations of the implantingprocedure and a clear plastic cover may be placed over the compartmentsto maintain sterility.

These and other aspects of the present application will be furtherappreciated upon consideration of the following Examples, which areintended to illustrate certain particular embodiments of the applicationbut are not intended to limit its scope, as defined by the claims.

EXAMPLE S

A predicate bone graft device contains demineralized bone matrix (DBM)particles in a sodium alginate/bovine collagen carrier. The product isprocessed such that terminal sterilization is required, which is appliedthrough exposure to E-beam radiation at 25-30 kGy. A new bone graftputty, which contains DBM fibers in a sodium alginate carrier was made.This study was undertaken to compare the new putty processed eitheraseptically (i.e. not requiring terminally irradiation), or followingE-beam irradiation, by assessing performance in a rat osteoinductivity(OI) model and in a rat two-level posterolateral spine fusion (PLF)model.

An aseptically processed (e.g., not requiring terminal irradiation)putty implant and an E-beam irradiated putty implant were compared toeach other by assessing performance in a rat osteoinductivity (OI) modeland in a rat two-level posterolateral spine fusion (PLF) model. Bothimplants contained the same ingredients such as DBM in fiber andparticle form disposed in a sodium alginate carrier.

Example 1 Manufacture of the Implants

1. Manufacture of Irradiated PLF Implants:

Implant putty samples were created in a biosafety cabinet by hand mixingcomponents shown in Table 1 below. 0.6 cc samples were loaded into 1 ccsyringes and packaged in foil pouches, and irradiated with E-beam at 27kilograys (kGy).

TABLE 1 Recipe for Implant Putty Composition Component by WeightDemineralized bone matrix (DBM) 25% Sterile filtered water 38% Sterilesodium alginate 6% Phosphate buffered saline (PBS) 32%

2. Manufacture of Aseptic PLF Implants:

The alginate and PBS components from Table 1 were manually mixed untilhomogeneous. The resulting gel was loaded into a 12 cc syringe andirradiated with E-beam at 27 kGy. A sterile environment was created in abiosafety cabinet, and the sterile gel was aseptically mixed with theother two components from Table 1 for a period of time. 0.6 cc sampleswere loaded into 1 cc syringes and packaged in pre-sterilized foilpouches.

3. Isolation of DBM from Irradiated and Aseptic OI Implants:

Leftover irradiated and aseptic materials from the PLF study implantmanufacturing were placed in separate 50 mL centrifuge tubes. 30 cc ofsterile water was added to each tube and implant materials were fullysuspended by vortexing the centrifuge tube. DBM was recovered from thesuspension by centrifugation at 3,800 revolutions per minute (RPM) for 5minutes. The process of resuspension and centrifugation was repeated twoadditional times such that the viscosity of supernatant water followingcentrifugation was indistinguishable from deionized (DI) water. 0.2 ccsamples were then loaded into ice syringes and packaged inpre-sterilized foil pouches.

Example 2 Rat 2-Level PLF Study

Twelve Athymic Nude rats were utilized in the study. The rats weredivided into two groups of six. Under general anesthesia and asepticconditions, surgical decortication of the dorsal surfaces of thetransverse processes of the 3rd, 4th and 5th lumbar vertebrae (L3-5) wasperformed in all animals. Test materials were placed on the paraspinalbeds bridging the decorticated transverse processes, followed bystandard wound closure. Spine radiography was performed immediatelyafter surgery, four weeks after surgery, and prior to necropsy at abouteight weeks after surgery. Appropriate post-operative analgesia andanimal care was provided. Animals were sacrificed eight weeks after thesurgery was performed. The lumbar spines were radiographed, explanted,palpated for motion in the operated segments during the necropsy andthen saved in 10% neutral buffered formalin. The results of the groupsare listed in FIG. 3.

It was concluded that irradiated implants fused 8 of 24 unilateralsegments (33%) as shown in FIG. 5, while aseptic implants fused 24 of 24unilateral segments (100%) by manual palpation and radiographically, asshown in FIG. 4. FIG. 4 illustrates an x-ray of rat subjects in group 1that were administered the injectable aseptic implant and their progressat 2 weeks, 4 weeks, and 8 weeks after implantation. FIG. 5 illustratesan x-ray of rat subjects from group 2 that were administered theirradiated moldable putty and their progress at 2 weeks, 4 weeks, and 8weeks after implantation. Greater consolidation and organization of newbone was observed radiographically in the aseptic group as opposed tothe irradiated treatment group.

Example 3 Rat OI Study

Four athymic male rats were used in this study with four implant sitesper test sample. Each animal received two intermuscular implants in theAdductor Brevis: Senthnentbranosus muscles. The samples were randomizedso that no animal received the same lot in both the left and rightimplant sites. The animals were anesthetized and prepared for surgery.Pockets were created using sharp and blunt dissection in the muscle.After the incision over the implant site was made, the sample was placedinto the muscle pocket, and then the pocket and skin were suturedclosed. The animals were in-life for 28 days and observed daily forabnormal general health status. At the end of the study duration,animals were sacrificed and the implants were removed. The tissues werefixed in 10% neutral buffered formalin prior to routine decalcificationand processed into paraffin blocks. At least four sections were cut fromthe block, mounted on slides and stained with hematoxylin and eosin(H&E). Slides were viewed under a microscope and interpreted by apathologist; the histopathology is semi-quantitative. A score wasassigned to each implant site as either positive or negative forevidence of new bone formation elements.

Results showed that an average OI score for the aseptic group (2.25) washigher than for the irradiated group (1.0).

In conclusion, aseptically processed putty implants demonstrated higherperformance in a rat 2-level posterolateral fusion model. Performance inthis model aligned with higher osteoinductivity of the DBM within theaseptic final product samples verse irradiated final product samples.

Example 4

An injectable putty was made that had similar handling to the predicateirradiated putty. The putty contained the following:

Composition Component Component by Weight Characteristic Demineralizedbone matrix (DBM) 28% DBM 50:50 Lyophilized fibers to particles. Fibershave a size 1 mm and above. Particles have a size of 100 microns to 1000microns. Sterile dry sodium alginate 6% Particles have a size of 100microns to 1000 microns. Phosphate buffered saline (PBS) 66%

The injectable putty has a higher percentage of diluent (e.g., PBS) andhaving DBM fibers in it leads to improved osteoconductivity compared tousing particles alone.

Example 5

A moldable implant was made that contained the following:

Composition Component Component by Weight Characteristic Demineralizedbone matrix (DBM) 30% 33:33:33 Lyophilized DBM fibers, chips, andparticles. Fibers have a size 1 mm and above. Particles have a size of100 microns to 1000 microns. Chips have a size of 1 micron to 4 microns.Sterile dry sodium alginate 14% Particles have a size of 100 microns to1000 microns Phosphate buffered saline (PBS) 56%

The moldable implant has a higher percentage of DBM in fibers, chips andparticles and lower percentage of diluent (e.g., PBS). DBM fibers leadto improved osteoconductivity compared to using particles alone.

Example 6 Rabbit PLF Study

1. Preclinical Study Model:

An un-instrumented single level rabbit posterolateral lumbar spinalfusion model was used in this study. Procedures were conducted onskeletally mature New Zealand White rabbits.

2. Specimen Preparation:

For Groups 1 and 2, the putty and moldable implants were provided insterile form in pre-packed syringes containing approximately 3 cc ofmaterial that were ready to use. The Group 1 putty contained lyophilizedDBM comprising a ratio of fibers to particles of 50:50, sterile drysodium alginate, PBS and sterile water. The Group 2 moldable implantcontained lyophilized DBM comprising a ratio of fibers, chips toparticles of 33:33:33, sterile dry sodium alginate, PBS and sterilewater. In Groups 3 and 4, 1.25-1.5 cc of test material putty andmoldable implants were discharged into a sterile weigh boat, combinedwith an equal volume of autograft morsels and thoroughly mixed. Themixture was loaded into an open barrel 3 cc syringe to measure thevolume prior to implantation. Therefore, the Group 3 material containeda combination product comprising the injectable putty of Group 1 and 50%autograft, and the Group 4 material contained a combination productcomprising the moldable implant of Group 2 and 50% autograft. For Group5, the predicate irradiated putty material containing demineralized bonematrix (DBM) particles in a sodium alginate/bovine collagen carrier, andthe control autograft of Group 6 was provided in a sterile, ready-to-useform.

3. Spine Fusion Procedure:

The dorsal surfaces of the transverse processes of L4 and L5 weredecorticated. Implants were deployed such that they were in contact withand spanned the distance between the decorticated L4 and L5 transverseprocesses bilaterally.

4. Autograft Harvest:

Autograft from the iliac crests was harvested bilaterally from Group 3,Group 4, and Group 6 animals. Harvested autograft was morselized usingRongeur forceps.

5. Necropsy:

Coronal x-rays were taken at necropsy. The lumbar spines were explanted,and the operated segments were palpated for motion. Operative sites weregraded as either “F” for fused or “M” for motion.

6. Results:

The results of this study are illustrated in FIGS. 6-8. Group 1 resultsshowed bilateral fusion in 3 out of 6 segments and unilateral fusion in6 out of 12 segments, as shown in FIG. 7. Group 2 results showedbilateral fusion in 0 out of 6 segments and unilateral fusion in 1 outof 12 segments, as shown in FIG. 8. Group 3 results showed bilateralfusion in 3 out of 6 segments and unilateral fusion in 6 out of 12segments. Group 4 results showed bilateral fusion in 1 out of 6 segmentsand unilateral fusion in 2 out of 12 segments. Group 5 results showedbilateral fusion in 0 out of 6 segments and unilateral fusion in 0 outof 12 segments. Group 6 results showed bilateral fusion in 3 out of 6segments and unilateral fusion in 6 out of 12 segments.

All patent and non-patent publications cited in this disclosure areincorporated herein in to the extent as if each of those patent andnon-patent publications was incorporated herein by reference in itsentirety. Further, even though the disclosure herein has been describedwith reference to particular examples and embodiments, it is to beunderstood that these examples and embodiments are merely illustrativeof the principles and applications of the present disclosure. It istherefore to be understood that numerous modifications may be made tothe illustrative embodiments and that other arrangements may be devisedwithout departing from the spirit and scope of the present disclosure asdefined by the following claims.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to various embodimentsdescribed herein without departing from the spirit or scope of theteachings herein. Thus, it is intended that various embodiments coverother modifications and variations of various embodiments within thescope of the present teachings.

What is claimed is:
 1. An injectable implant configured to fit at ornear a bone defect to promote bone growth, the injectable implantcomprising lyophilized demineralized bone matrix (DBM) being in fiberand particle forms; alginate; and a liquid carrier, wherein the DBM isin an amount of about 20 wt. % to about 40 wt. % of a total weight ofthe injectable implant, the alginate is in an amount of from about 1 wt.% to about 10 wt. % of the total weight of the injectable implant, andthe liquid carrier is in an amount from about 50 wt. % to about 70 wt. %of the total weight of the injectable implant.
 2. The injectable implantof claim 1, wherein (i) the defect is a bone void and the injectableimplant is a bone void filler that is an injectable putty form; or (ii)the liquid carrier is phosphate buffered saline (PBS).
 3. The injectableimplant of claim 2, wherein the DBM is in an amount of about 28 wt. %based on the total weight of the injectable implant, the alginate is inan amount of about 6 wt. % based on the total weight of the injectableimplant, and the phosphate buffered saline is in an amount of about 66wt. % based on the total weight of the injectable implant.
 4. Theinjectable implant of claim 1, wherein the fiber form has a size of fromabout 1 to about 7 mm, and the particle form has a size of from about100 microns to about 1000 microns.
 5. The injectable implant of claim 1,wherein the injectable implant is an aseptically processed injectableimplant, the injectable implant further comprises sterile water, and isin a putty form.
 6. The injectable implant of claim 1, wherein the ratioof fibers and particle forms is 50:50.
 7. The injectable implant ofclaim 1, wherein the alginate is a sterile sodium alginate powder andthe alginate has a particle size of from about 100 to about 1,000microns.
 8. The injectable implant of claim 1, wherein the injectableimplant does not contain collagen.
 9. The injectable implant of claim 1,wherein the injectable implant further comprises at least one viscosityenhancing agent, a biodegradable polymer, a mineral particle or atherapeutic agent.
 10. A moldable implant configured to fit at or near abone defect to promote bone growth, the moldable implant comprisinglyophilized demineralized bone matrix (DBM) being in fiber, particle andchip forms, the DBM being in the moldable implant in an amount of about15 wt. % to about 40 wt. % based on a total weight of the moldableimplant; and the moldable implant comprising an alginate in an amount offrom about 3 wt. % to about 20 wt. % based on the total weight of themoldable implant.
 11. The moldable implant of claim 10, wherein themoldable implant further comprises an aqueous carrier comprisingphosphate buffered saline in an amount from about 40 wt. % to about 60wt. % based on the total weight of the moldable implant and sterilefiltered water.
 12. The moldable implant of claim 10, wherein the defectis a bone void and the moldable implant is a bone void filler being in aputty form.
 13. The moldable implant of claim 11, wherein the DBM is inan amount of about 30 wt. % based on the total weight of the moldableimplant, the alginate is in an amount of about 14 wt. % based on thetotal weight of the moldable implant, and the phosphate buffered salineis in an amount of about 54 wt. % based on the total weight of themoldable implant.
 14. The moldable implant of claim 10, wherein thefiber form has a size of from about 1 to about 7 mm, the particle formhas a size of from about 100 microns to about 1000 microns, and the chipform has a size of from about 1 to about 4 mm.
 15. The moldable implantof claim 10, wherein the moldable implant is an aseptically processedimplant and is in a putty form.
 16. The moldable implant of claim 10,wherein the fiber, particle and chip forms are fully demineralized, andthe ratio of fiber, particle and chip forms is 33:33:33.
 17. Themoldable implant of claim 10, wherein the alginate is a sterile sodiumalginate powder and the alginate has a particle size of from about 100to about 1,000 microns.
 18. The moldable implant of claim 10, whereinthe moldable implant does not contain collagen.
 19. The moldable implantof claim 10, wherein the moldable implant further comprises at least oneviscosity enhancing agent, a biodegradable polymer, a mineral particle,or a therapeutic agent.
 20. A method of making an injectable or moldableimplant, the method comprising mixing lyophilized demineralized bonematrix (DBM) being in fiber and particle forms in an amount of about 28%based on a total weight of the implant, or DBM in fiber, particle andchip forms in an amount of about 30% based on the total weight of theimplant with an aqueous liquid comprising an alginate so as to uniformlydistribute the DBM within the alginate to form the injectable ormoldable implant.